FIG. 4.

HBZ displaces CREB from the viral CRE in vitro. (A) HBZ-bZIP reduces CREB and CREB-bZIP binding to the viral CRE. Gel shift assays were used to analyze protein-DNA interactions. Binding reactions in lanes 1 to 11 contained 2.3 fmol of ³²P-labeled viral CRE 3 probe with 0.16 pmol of CREB-bZIP (lanes 2 to 6) or 0.16 pmol of full-length CREB (lanes 7 to 11). Binding reactions in lanes 12 to 17 contained 3 fmol of ³²P-labeled UAS probe (lanes 12 to 17) with 0.35 pmol of Gal4-VP16 (lanes 13 to 17). The reactions shown in lanes 3 to 6 and 8 to 11 also contained increasing amounts of HBZ-bZIP (0.02, 0.04, 0.08, and 0.16 pmol) and in lanes 14 to 17 (0.044, 0.09, 0.175, and 0.35 pmol). Positions of the free probe and relevant protein-DNA complexes are labeled. The minor bands that migrated below the major CREB homodimer are truncated forms of CREB present in the purified preparation. The slower-migrating complexes in CREB-bZIP lanes are CREB-bZIP aggregates typically observed with high concentrations of protein (represented by asterisks). (B) HBZ-ΔbZIP does not reduce CREB and CREB-bZIP binding to the viral CRE. Binding reactions contained 6.8 fmol of ³²P-labeled viral CRE 3 probe (lanes 1 to 12) with 0.08 pmol of CREB (lanes 2 to 6) and 0.12 pmol of CREB-bZIP (lanes 8 to 12). The reaction shown in lane 3 also contained 0.8 pmol of HBZ-bZIP, and the reactions shown in lanes 4 to 6 contained increasing amounts of HBZ-ΔbZIP (0.8, 1.6, and 2.4 pmol). The reaction shown in lane 9 also contained 1.2 pmol of HBZ-bZIP, and the reactions shown in lanes 10 to 12 contained increasing amounts of HBZ-ΔbZIP (1.2, 1.8, and 2.4 pmol). (C) Specificity of CREB and CREB-bZIP binding to the viral CRE. Binding reactions contained 6.8 fmol of ³²P-labeled viral CRE 3 probe (lanes 1 to 8) with 0.2 pmol of CREB (lanes 2 and 4) and 0.5 pmol of CREB-bZIP (lanes 5 to 7). An antibody directed against the bZIP domain of CREB (500 ng, lanes 3 and 6) or an antibody against histone H3 (500 ng, lanes 4 and 7) were added in the reactions. (D) Purified proteins used in gel shift assays. Purified CREB (lane 2), CREB-bZIP (lane 3), and HBZ-ΔbZIP (lane 4) are shown by Coomassie blue staining after SDS-PAGE. Protein standards (in kilodaltons) are indicated in lane 1. (E) HBZ-bZIP does not displace CREB-bZIP prebound to the viral CRE. Binding reactions contained 6 fmol of ³²P-labeled viral CRE 3 probe with 0.16 pmol of the CREB-bZIP (lanes 2 to 13). Increasing amounts of HBZ-bZIP (0.04, 0.08, 0.16, 0.32, and 0.64 pmol) were added at the same time as CREB and DNA (lanes 3 to 7) or 30 min after CREB and DNA (lanes 9 to 13). The positions of the free probe and relevant protein-DNA complexes are labeled. The slower-migrating complexes in CREB-bZIP lanes are CREB-bZIP aggregates typically observed with high concentrations of CREB-bZIP (represented by asterisks).